Electropipettor and compensation means for electrophoretic bias

ABSTRACT

A channel ( 140 ) is divised into portions ( 142, 144 ). The sidewalls of each channel portion ( 142, 144 ) have surface charges of opposite polarity. The two channel portions ( 142, 144 ) are physically connected together by a salt bridge ( 133 ), such as a glass frit or gel layer. The salt bridge ( 133 ) separates the fluids in channel ( 140 ) from an ionic fluid reservoir ( 135 ). To impart electroosmotic and electrophoretic forces along the channel ( 140 ) between parts A and B, respectively. Additionally, a third electrode ( 137 ) is placed in the reservoir ( 135 ).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 08/760,446, filed Dec. 6, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/671,986,filed Jun. 28, 1996, all of which are incorporated herein by referencein their entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] There has been a growing interest in the manufacture and use ofmicrofluidic systems for the acquisition of chemical and biochemicalinformation. Techniques commonly associated with the semiconductorelectronics industry, such as photolithography, wet chemical etching,etc., are being used in the fabrication of these microfluidic systems.The term, “microfluidic”, refers to a system or device having channelsand chambers which are generally fabricated at the micron or submicronscale, e.g., having at least one cross-sectional dimension in the rangeof from about 0.1 μm to about 500 μm. Early discussions of the use ofplanar chip technology for the fabrication of microfluidic systems areprovided in Manz et al., Trends in Anal. Chem. (1990) 10(5):144-149 andManz et al., Avd. in Chromatog. (1993) 33:1-66, which describe thefabrication of such fluidic devices and particularly microcapillarydevices, in silicon and glass substrates.

[0003] Applications of microfluidic systems are myriad. For example,International Patent Appln. WO 96/04547, published Feb. 15, 1996,describes the use of microfluidic systems for capillary electrophoresis,liquid chromatography, flow injection analysis, and chemical reactionand synthesis. U.S. patent application Ser. No. 08/671,987, filed Jun.28, 1996, and incorporated herein by reference, discloses wide rangingapplications of microfluidic systems in rapidly assaying large number ofcompounds for their effects on chemical, and preferably, biochemicalsystems. The phrase, “biochemical system,” generally refers to achemical interaction which involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signaling and other reactions. Biochemicalsystems of particular interest include, e.g., receptor-ligandinteractions, enzyme-substrate interactions, cellular signalingpathways, transport reactions involving model barrier systems (e.g.,cells or membrane fractions) for bioavailability screening, and avariety of other general systems.

[0004] Many methods have been described for the transport and directionof fluids, e.g., samples, analytes, buffers and reagents, within thesemicrofluidic systems or devices. One method moves fluids withinmicrofabricated devices by mechanical micropumps and valves within thedevice. See, Published U.K. Patent Application No. 2 248 891 (Oct, 18,1990), Published European Patent Application No. 568 902 (May 2, 1992),U.S. Pat. Nos. 5,271,724 (Aug. 21, 1991) and 5,277,556 (Jul. 3, 1991).See also, U.S. Pat. No. 5,171,132 (Dec. 21, 1990) to Miyazaki et al.Another method uses acoustic energy to move fluid samples within devicesby the effects of acoustic streaming. See, Published PCT Application No.94/05414 to Northrup and White. A straightforward method appliesexternal pressure to move fluids within the device. See, e.g., thediscussion in U.S. Pat. No. 5,304,487 to Wilding et al.

[0005] Still another method uses electric fields to move fluid materialsthrough the channels of the microfluidic system. See, e.g., PublishedEuropean Patent Application No. 376 611 (Dec. 30, 1988) to Kovacs,Harrison et al., Anal. Chem. (1992) 64:1926-1932 and Manz et al. J.Chromatog. (1992) 593:253-258, U.S. Pat. No. 5,126,022 to Soane.Electrokinetic forces have the advantages of direct control, fastresponse and simplicity. However, there are still some disadvantages.For maximum efficiency, it is desirable that the subject materials betransported as closely together as possible. Nonetheless, the materialsshould be transported without cross-contamination from other transportedmaterials. Further, the materials in one state at one location in amicrofluidic system should remain in the same state after being moved toanother location in the microfluidic system. These conditions permit thetesting, analysis and reaction of the compound materials to becontrolled, when and where as desired.

[0006] In a microfluidic system in which the materials are moved byelectrokinetic forces, the charged molecules and ions in the subjectmaterial regions and in the regions separating these subject materialregions are subjected to various electric fields to effect fluid flow.

[0007] Upon application of these electric fields, however; differentlycharged species within the subject material will exhibit differentelectrophoretic mobilities, i.e., positively charged species will moveat a different rate than negatively charged species. In the past, theseparation of different species within a sample that was subjected to anelectric field was not considered a problem, but was, in fact, thedesired result, e.g., in capillary electrophoresis. However, wheresimple fluid transport is desired, these varied mobilities can result inan undesirable alteration or “electrophoretic bias” in the subjectmaterial.

[0008] Without consideration and measures to avoid cross-contamination,the microfluidic system must either widely separate the subjectmaterials, or, in the worst case, move the materials one at a timethrough the system. In either case, efficiency of the microfluidicsystem is markedly reduced. Furthermore, if the state of the transportedmaterials cannot be maintained in transport, then many applicationswhich require the materials to arrive at a location unchanged must beavoided.

[0009] The present invention solves or substantially mitigates theseproblems of electrokinetic transport. With the present invention,microfluidic systems can move materials efficiently and withoutundesired change in the transported materials. The present inventionpresents a high throughput microfluidic system having direct, fast andstraightforward control over the movement of materials through thechannels of the microfluidic system with a wide range of applications,such as in the fields of chemistry, biochemistry, biotechnology,molecular biology and numerous other fields.

SUMMARY OF THE INVENTION

[0010] The present invention provides for a microfluidic system whichelectroosmotically moves subject material along channels in fluid slugs,also termed “subject material regions,” from a first point to a secondpoint in the microfluidic system. A first spacer region of high ionicconcentration contacts each subject material region on at least one sideand second spacer regions of low ionic concentration are arranged withthe subject material regions of subject material and first or high ionicconcentration spacer regions so that at least one low ionicconcentration region is always between the first and second points toensure that most of the voltage drop and resulting electric fieldbetween the two points is across the low ionic concentration region.

[0011] The present invention also provides for a electropipettor whichis compatible with a microfluidic system which moves subject materialswith electroosmotic forces. The electropipettor has a capillary having achannel. An electrode is attached along the outside length of thecapillary and terminates in a electrode ring at the end of thecapillary. By manipulating the voltages on the electrode and theelectrode at a target reservoir to which the channel is fluidlyconnected when the end of the capillary is placed into a materialsource, materials are electrokinetically introduced into the channel. Atrain of subject material regions, high and low ionic concentrationbuffer or spacer regions can be created in the channel for easyintroduction into the microfluidic system.

[0012] The present invention further compensates for electrophoreticbias as the subject materials are electrokinetically transported alongthe channels of a microfluidic system. In one embodiment a channelbetween two points of the microfluidic system has two portions withsidewalls of opposite surface charges. An electrode is placed betweenthe two portions. With the voltages at the two points substantiallyequal and the middle electrode between the two portions set differently,electrophoretic forces are in opposite directions in the two portions,while electroosmotic forces are in the same direction. As subjectmaterial is transported from one point to the other, electrophoreticbias is compensated for, while electroosmotic forces move the fluidmaterials through the channel.

[0013] In another embodiment a chamber is formed at the intersection ofchannels of a microfluidic system. The chamber has sidewalls connectingthe sidewalls of the intersecting channels. When a subject materialregion is diverted from one channel into another channel at theintersection, the chamber sidewalls funnel the subject material regioninto the second channel. The width of the second channel is such thatdiffusion mixes any subject material which had been electrophoreticallybiased in the subject material region as it traveled along the firstchannel.

[0014] In still a further embodiment, the present invention provides amicrofluidic system and method of using that system for controllablydelivering a fluid stream within a microfluidic device having at leasttwo intersecting channels. The system includes a substrate having the atleast two intersecting channels disposed therein. In this aspect, theone of the channels is deeper than the other channel. The system alsoincludes an electroosmotic fluid direction system. The system isparticularly useful where the fluid stream comprises at least two fluidregions having different ionic strengths.

[0015] The present invention also provides a sampling system using theelectropipettor of the invention. The sampling system includes a samplesubstrate, which has a plurality of different samples immobilizedthereon. Also included is a translation system for moving theelectropipettor relative to said sample substrate.

[0016] The invention as hereinbefore described may be put into aplurality of different uses, which are themselves inventive, forexample, as follows:

[0017] The use of a substrate having a channel, in transporting at leasta first subject material from at least a first location to a secondlocation along the channel, utilizing at least one region of low ionicconcentration which is transported along the channel due to an appliedvoltage.

[0018] A use of the aforementioned invention, in which the ionicconcentration of the one region is substantially lower than that of thesubject material.

[0019] A use of the aforementioned invention, wherein a plurality ofsubject materials are transported, separated by high ionic concentrationspacer regions.

[0020] The use of a substrate having a channel along which at least afirst subject material may be transported, in electrophoretic biascompensation, the channel being divided into a first and a secondportion, in which the wall or walls of the channel are oppositelycharged, such that electrophoretic bias on the at least first subjectmaterial due to transportation in the first portion is substantiallycompensated for by electrophoretic bias due to transport in the secondportion.

[0021] A use of the aforementioned invention in which a first electrodeis located at a remote end of the first portion, a second electrode islocated at the intersection between the portions and a third electrodeis located at a remote end of the second portion.

[0022] A use of the aforementioned invention, in which the substrate isa microfluidic system.

[0023] A use of the aforementioned invention in which the substrate isan electropipettor.

[0024] A use of the aforementioned invention, in which theelectropipettor has a main channel for transportation of the subjectmaterial and at least one further channel fluidly connected to the mainchannel from which a further material to be transported along the mainchannel is obtained.

[0025] A use of the aforementioned invention, in which the furthermaterial is drawn into the main channel as a buffer region between eachof a plurality of separate subject materials.

[0026] The use of a microfluidic system having at least a first and asecond fluid channel which intersect, in optimizing flow conditions, thechannels having different depths.

[0027] A use of the aforementioned invention in which one channel isbetween 2 to 10 times deeper than the other channel.

[0028] The use of a microfluidic system having a first channel and asecond channel intersecting the first channel, in electrophoreticcompensation, the intersection between the channels being shaped suchthat a fluid being transported along the first channel towards thesecond channel is mixed at the intersection and any electrophoretic biasin the fluid is dissipated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 shows a schematic illustration of one embodiment of amicrofluidic system;

[0030]FIG. 2A illustrates an arrangement of fluid regions traveling in achannel of the microfluidic system of FIG. 1, according to oneembodiment of the present invention; Figure B is a scaled drawing ofanother arrangement of different fluid regions traveling in a channel ofthe microfluidic system according to the present invention;

[0031]FIG. 3A is another arrangement with high ionic concentrationspacer regions before a subject material region traveling within achannel of the microfluidic system; FIG. 3B shows an arrangement withhigh ionic concentration spacer regions after a subject material regiontraveling in a channel of the microfluidic system;

[0032]FIG. 4A is a schematic diagram of one embodiment of aelectropipettor according to the present invention; FIG. 4B is aschematic diagram of another electropipettor according to the presentinvention;

[0033]FIG. 5 is a schematic diagram of a channel having portions withoppositely charged sidewalls in a microfluidic system, according to thepresent invention; and

[0034] FIGS. 6A-6D illustrate the mixing action of funneling sidewallsat the intersection of channels in a microfluidic system, according tothe present invention.

[0035]FIG. 7A shows the results of three injections of a sample fluidmade up of two oppositely charged chemical species in a low salt buffer,into a capillary filled with low salt buffer. FIG. 7B shows the resultsof three sample injections where the sample is in high salt buffer, highsalt buffer fluids were injected at either end of the sample region tofunction as guard bands, and the sample/guard bands were run in a lowsalt buffer filled capillary. FIG. 7C shows the results of three sampleinjections similar to that of FIG. 7B except that the size of the lowsalt spacer region between the sample/high salt spacers (guard bands) isreduced, allowing partial resolution of the species within the sample,without allowing the sample elements to compromise subsequent orprevious samples.

[0036]FIG. 8 shows a schematic illustration of an electropipettor foruse with a sampling system using samples immobilized, e.g., dried, on asubstrate sheet or matrix.

[0037]FIG. 9A is a plot of fluorescence versus time which illustratesthe movement of a sample fluid made up of test chemical species which isperiodically injected into, and moved through, an electropipettor,according to the present invention. FIG. 9B is another plot which showsthe movement of the sample fluid with the chemical species through amicrofluidic substrate which is connected to the electropipettor, underdifferent parameters. FIG. 9C is a plot which illustrates the movementof the sample fluid and chemical species through an electropipettorformed from an air abraded substrate.

[0038]FIG. 10 is a plot which again illustrates the movement of achemical species in a sample fluid which has been periodically injectedinto an electropipettor, according to the present invention. In thisexperiment, the species is a small molecule compound.

DETAILED DESCRIPTION OF THE INVENTION

[0039] I. General Organization of a Microfluidic System

[0040]FIG. 1 discloses a representative diagram of an exemplarymicrofluidic system 100 according to the present invention. As shown,the overall device 100 is fabricated in a planar substrate 102. Suitablesubstrate materials are generally selected based upon theircompatibility with the conditions present in the particular operation tobe performed by the device. Such conditions can include extremes of pH,temperature, ionic concentration, and application of electrical fields.Additionally, substrate materials are also selected for their inertnessto critical components of an analysis or synthesis to be carried out bythe system.

[0041] Useful substrate materials include, e.g., glass, quartz andsilicon, as well as polymeric substrates, e.g., plastics. In the case ofconductive or semiconductive substrates, there should be an insulatinglayer on the substrate. This is particularly important where the deviceincorporates electrical elements, e.g., electrical fluid directionsystems, sensors and the like, or uses electroosmotic forces to movematerials about the system, as discussed below. In the case of polymericsubstrates, the substrate materials may be rigid, semi-rigid, ornon-rigid, opaque, semi-opaque or transparent, depending upon the usefor which they are intended. For example, devices which include anoptical or visual detection element, will generally be fabricated, atleast in part, from transparent materials to allow, or at least,facilitate that detection. Alternatively, transparent windows of, e.g.,glass or quartz, may be incorporated into the device for these types ofdetection elements. Additionally, the polymeric materials may havelinear or branched backbones, and may be crosslinked or non-crosslinked.Examples of particularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC),polystyrene, polysulfone, polycarbonate and the like.

[0042] The system shown in FIG. 1 includes a series of channels 110,112, 114 and 116 fabricated into the surface of the substrate 102. Asdiscussed in the definition of “microfluidic,” these channels typicallyhave very small cross sectional dimensions, preferably in the range offrom about 0.1 μm to about 100 μm. For the particular applicationsdiscussed below, channels with depths of about 10 μm and widths of about60 μm work effectively, though deviations from these dimensions are alsopossible.

[0043] Manufacturing of these channels and other microscale elementsinto the surface of the substrate 102 may be carried out by any numberof microfabrication techniques that are well known in the art. Forexample, lithographic techniques may be employed in fabricating glass,quartz or silicon substrates, for example, with methods well known inthe semiconductor manufacturing industries. Photolithographic masking,plasma or wet etching and other semiconductor processing technologiesdefine microscale elements in and on substrate surfaces. Alternatively,micromachining methods, such as laser drilling, micromilling and thelike, may be employed. Similarly, for polymeric substrates, well knownmanufacturing techniques may also be used. These techniques includeinjection molding techniques or stamp molding methods where largenumbers of substrates may be produced using, e.g., rolling stamps toproduce large sheets of microscale substrates, or polymer microcastingtechniques where the substrate is polymerized within a microfabricatedmold.

[0044] Besides the substrate 102, the microfluidic system includes anadditional planar element (not shown) which overlays the channeledsubstrate 102 to enclose and fluidly seal the various channels to formconduits. The planar cover element may be attached to the substrate by avariety of means, including, e.g., thermal bonding, adhesives or, in thecase of glass, or semi-rigid and non-rigid polymeric substrates, anatural adhesion between the two components. The planar cover elementmay additionally be provided with access ports and/or reservoirs forintroducing the various fluid elements needed for a particular screen.

[0045] The system 100 shown in FIG. 1 also includes reservoirs 104, 106and 108, which are disposed and fluidly connected at the ends of thechannels 114, 116 and 110 respectively. As shown, sample channel 112, isused to introduce a plurality of different subject materials into thedevice. As such, the channel 112 is fluidly connected to a source oflarge numbers of separate subject materials which are individuallyintroduced into the sample channel 112 and subsequently into anotherchannel 110. As shown, the channel 110 is used for analyzing the subjectmaterials by electrophoresis. It should be noted that the term, “subjectmaterials,” simply refers to the material, such as a chemical orbiological compound, of interest. Subject compounds may include a widevariety of different compounds, including chemical compounds, mixturesof chemical compounds, e.g., polysaccharides, small organic or inorganicmolecules, biological macromolecules, e.g., peptides, proteins, nucleicacids, or extracts made from biological materials, such as bacteria,plants, fungi, or animal cells or tissues, naturally occurring orsynthetic compositions.

[0046] The system 100 moves materials through the channels 110, 112, 114and 116 by electrokinetic forces which are provided by a voltagecontroller that is capable of applying selectable voltage levels,simultaneously, to each of the reservoirs, including ground. Such avoltage controller can be implemented using multiple voltage dividersand multiple relays to obtain the selectable voltage levels.Alternatively, multiple independent voltage sources may be used. Thevoltage controller is electrically connected to each of the reservoirsvia an electrode positioned or fabricated within each of the pluralityof reservoirs. See, for example, published International PatentApplication No. WO 96/04547 to Ramsey, which is incorporated herein byreference in its entirety for all purposes.

[0047] II. Electrokinetic Transport

[0048] A. Generally

[0049] The electrokinetic forces on the fluid materials in the channelsof the system 100 may be separated into electroosmotic forces andelectrophoretic forces. The fluid control systems used in the system ofthe present invention employ electroosmotic force to move, direct andmix fluids in the various channels and reaction chambers present on thesurface of the substrate 102. In brief, when an appropriate fluid isplaced in a channel or other fluid conduit having functional groupspresent at the surface, those groups can ionize. Where the surface ofthe channel includes hydroxyl functional groups at the surface, forexample, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface possesses a net negativecharge, whereas the fluid possesses an excess of protons or positivecharge, particularly localized near the interface between the channelsurface and the fluid.

[0050] By applying an electric field across the length of the channel,cations flow toward the negative electrode. Movement of the positivelycharged species in the fluid pulls the solvent with them. The steadystate velocity of this fluid movement is generally given by theequation:$v = \frac{\varepsilon \quad \xi \quad E}{4\pi \quad \eta}$

[0051] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. Thus, as can be easilyseen from this equation, the solvent velocity is directly proportionalto the zeta potential and the applied field.

[0052] Besides electroosmotic forces, there are also electrophoreticforces which affect charged molecules as they move through the channelsof the system 100. In the transport of subject materials from one pointto another point in the system 100, it is often desirable for thecomposition of the subject materials to remain unaffected in thetransport, i.e., that the subject materials are not electrophoreticallydifferentiated in the transport.

[0053] In accordance with the present invention, the subject materialsare moved about the channels as slugs of fluid (hereafter termed“subject material regions”), which have a high ionic concentration tominimize electrophoretic forces on the subject materials within theseparticular regions. To minimize the effect of electrophoretic forceswithin the subject material regions, regions of spacer fluids (“firstspacer regions”) are placed on either side of a slug. These first spacerregions have a high ionic concentration to minimize the electric fieldsin these regions, as explained below, so that subject materials areessentially unaffected by the transport from one location to anotherlocation in a microfluidic system. The subject materials are transportedthrough the representative channels 110, 112, 114, 116 of the system 100in regions of certain ionic strengths, together with other regions ofionic strengths varying from those regions bearing the subjectmaterials.

[0054] A specific arrangement is illustrated in FIG. 2A, whichillustrates subject material regions 200 being transported from point Ato point B along a channel of the microfluidic system 100. In eitherside of the subject material regions 200 are first spacer regions 201 ofhigh ionic strength fluid. Additionally, second spacer regions 202 oflow ionic concentration fluid periodically separate arrangements ofsubject material regions 200 and first spacer regions 201. Being of lowionic concentration, most of the voltage drop between points A and Boccurs across these second spacer regions 202. The second or lowconcentration spacer regions 202 are interspersed between thearrangements of subject material region 200 and first spacer region 201such that, as the subject material regions 200 and the first spacerregions 201 are electroosmotically pumped through the channel, there isalways at least one second or low ionic concentration spacer region 202between the points A and B. This ensures that most of the voltage dropoccurs in the second spacer region 202, rather than across the subjectmaterial region 200 and first spacer regions 201. Stated differently,the electric field between points A and B is concentrated in the secondspacer region 202 and the subject material regions 200 and first spacerregions 201 experience low electric fields (and low electrophoreticforces). Thus, depending upon the relative ionic concentrations in thesubject material regions 200, first spacer regions 201 and second or lowionic concentration spacer regions 202, other arrangements of thesesubject material regions 200, and first and second spacer regions 201and 202 can be made.

[0055] For example, FIG. 2B illustrates an arrangement in which a secondor low ionic concentration spacer region 202 is regularly spaced betweeneach combination of first spacer region 201/subject material region200/first spacer region 201. Such an arrangement ensures that there isalways at least one second or low concentration spacer region 202between points A and B. Furthermore, the drawings are drawn to scale toillustrate the relative lengths of a possible combination of subjectmaterial region 200, first or high concentration spacer region 201 andsecond or low concentration spacer region 202. In the example of FIG.2B, the subject material region 200 holds the subject material in a highionic concentration of 150 mM of NaCl. The subject material region 200is 1 mm long in the channel. The two first spacer regions 201 have ionicconcentrations of 150 mM of NaCl. Each first spacer region 201 is 1 mmlong. The second spacer region 202 is 2 mm and has an ionicconcentration of 5 mM of borate buffer. This particular configuration isdesigned to maintain a rapidly electrophoresing compound in the subjectmaterial region 200 and buffer regions 201 while the compound travelsthrough the channels of the microfluidic system. For example, usingthese methods, a subject material region containing, e.g., benzoic acid,can be flowed through a microfluidic system for upwards of 72 secondswithout suffering excessive electrophoretic bias.

[0056] Stated more generally, the velocity of fluid flow, vεoF, throughthe channels of the microfluidic system can be determined and, bymeasurement, it is possible to determine the total distance, l_(T),which a subject matter molecule is to travel through the channels. Thusthe transit time, t_(Tr), for the subject matter molecule to travel thetotal distance is:

t _(Tr) =l _(T) /V _(εoF)

[0057] To contain a subject matter molecule x within the first spacerregion 201 next to the subject material region 200, the length of thefirst spacer region 201, l_(g). should be greater than theelectrophoretic velocity of the subject matter molecule x in the firstspacer region 201, v_(gx), multiplied by the transit time:

l _(g)>(v _(gx))(t _(Tr))

[0058] Since the electrophoretic velocity is proportional to theelectric field in the first spacer region 201, the present inventionallows control over v_(gx) so that the subject materials can becontained in transport through the microfluidic system channels.

[0059] In the arrangements in FIGS. 2A and 2B, the first or high ionicconcentration spacer regions 201 help maintain the position of thesubject materials in the vicinity of its subject material region 200. Nomatter what the polarity of the charges of the subject material, thefirst spacer regions 201 on either side of the subject material region200 ensures that any subject material leaving the subject materialregion 200 is only subject to a low electric field due to the relativehigh ionic concentrations in the first spacer regions 201. If thepolarity of the subject material is known, then the direction of theelectrophoretic force on the molecules of the subject material is alsoknown.

[0060]FIG. 3A illustrates an example where charges of the subjectmaterial in all the subject material regions 200 are such that theelectrophoretic force on the subject material molecules are in the samedirection as the direction of the electroosmotic flow. Hence the firstspacer regions 201 precede the subject material regions 200 in thedirection of flow. There are no first spacer regions 201 following thesubject material regions 200 because the electrophoretic force keeps thesubject material from escaping the subject material region 200 in thatdirection. By eliminating one-half of the first spacer regions 201, moresubject material regions 200 with their subject material can be carriedper channel length. This enhances the transportation efficiency of themicrofluidic system. The second or low ionic concentration spacerregions 202 are arranged with respect to the subject material regions200 and the first or high ionic concentration spacer regions 201 so thathigh electric fields fall in the second spacer regions 202 and theelectric fields (and electrophoretic forces) in the subject materialregions 200 and first spacer regions 201 are kept low.

[0061] In FIG. 3B the first spacer regions 201 follow the subjectmaterial regions 200 in the direction of the electroosmotic flow. Inthis example, the charges of the subject material in all the subjectmaterial regions 200 are such that the electrophoretic force on thesubject matter molecules are in the opposite direction as the directionof the electroosmotic flow. Hence the subject material may escape theconfines of its subject material region, in effect, being left behind byits subject material region 200. The first spacer regions 201 followingthe subject material regions 200 keep the subject material frommigrating too far from its subject material region 200. Likewise, thesecond or low ionic concentration spacer regions 202 are arranged withthe subject material regions 200 and the first or high ionicconcentration spacer regions 201 so that high electric fields fall inthe second spacer regions 202 and the electric fields in the subjectmaterial regions 200 and first spacer regions 201 are kept low.

[0062] Various high and low ionic strength solutions are selected toproduce a solution having a desired electrical conductivity for thefirst and second spacer regions 201 and 202. The specific ions thatimpart electrical conductivity to the solution maybe derived frominorganic salts (such as NaCl, KI, CaCl₂, FeF₃, (NH₄)₂SO₄ and so forth),organic salts (such as pyridinium benzoate, benzalkonium laurate), ormixed inorganic/organic salts (such as sodium benzoate, sodiumdeoxylsulfate, benzylaminehydrochloride). These ions are also selectedto be compatible with the chemical reactions, separations, etc. to becarried out in the microfluidic system. In addition to aqueous solvents,mixtures of aqueous/organic solvents, such as low concentrations of DMSOin water, may be used to assist in the solubilization of the subjectmatter molecules. Mixtures of organic solvents, such as CHCl₃:MeOH, maybe also used for the purpose of accelerating assays for phospholipaseactivity, for example.

[0063] Generally, when aqueous solvents are used, solution conductivityis adjusted using inorganic ions. When less polar solvents are used,organic or mixed inorganic/organic ions are typically used. In caseswhere two immiscible solvents may be simultaneously present (e.g., waterand a hydrocarbon such as decane) such that electrical current must flowfrom one into the other, ionophores (e.g., valinomycin, nonactin,various crown ethers, etc.) and their appropriate ions may be used toconduct current through the non-polar solvent.

[0064] B. Electrokinetic Control of Pressure Based Flow

[0065] In the electrokinetic flow systems described herein, the presenceof differentially mobile fluids (e.g., having a different electrokineticmobility in the particular system) in a channel may result in multipledifferent pressures being present along the length of a channel in thesystem. For example, these electrokinetic flow systems typically employa series of regions of low and high ionic concentration fluids (e.g.,first and second spacer regions and subject material regions of subjectmaterial) in a given channel to effect electroosmotic flow, while at thesame time, preventing effects of electrophoretic bias within a subjectmaterial containing subject material region. As the low ionicconcentration regions within the channel tend to drop the most appliedvoltage across their length, they will tend to push the fluids through achannel. Conversely, high ionic concentration fluid regions within thechannel provide relatively little voltage drop across their lengths, andtend to slow down fluid flow due to viscous drag.

[0066] As a result of these pushing and dragging effects, pressurevariations can generally be produced along the length of a fluid filledchannel. The highest pressure is typically found at the front or leadingedge of the low ionic concentration regions (e.g., the second spacerregions), while the lowest pressure is typically found at the trailingor back edge of these low ionic strength fluid regions.

[0067] While these pressure differentials are largely irrelevant instraight channel systems, their effects can result in reduced controlover fluid direction and manipulation in microfluidic devices thatemploy intersecting channel arrangements, i.e., the systems described inU.S. patent application Ser. No. 08/671,987, previously incorporated byreference. For example, where a second channel is configured tointersect a first channel which contains fluid regions of varying ionicstrength, the pressure fluctuations described above can cause fluid toflow in and out of the intersecting second channel as these differentfluid regions move past the intersection. This fluctuating flow couldpotentially, significantly disturb the quantitative electroosmoticallydriven flow of fluids from the second channel, and/or perturb thevarious fluid regions within the channel.

[0068] By reducing the depth of the intersecting channel, e.g., thesecond channel, relative to the first or main channel, the fluctuationsin fluid flow can be substantially eliminated. In particular, inelectroosmotic fluid propulsion or direction, for a given voltagegradient, the rate of flow (volume/time) generally varies as thereciprocal of the depth of the channel for channels having an aspectratio of >10 (width:depth). With some minor, inconsequential error forthe calculation, this general ratio also holds true for lower aspectratios, e.g., aspect ratios >5. Conversely, the pressure induced flowfor the same channel will vary as the third power of the reciprocal ofthe channel depth. Thus, the pressure build-up in a channel due to thesimultaneous presence of fluid regions of differing ionic strength willvary as the square of the reciprocal of the channel depth.

[0069] Accordingly, by decreasing the depth of the intersecting secondchannel relative to the depth of the first or main channel by a factorof X, one can significantly reduce the pressure induced flow, e.g., by afactor of X³, while only slightly reducing the electroosmoticallyinduced flow, e.g., by a factor of X. For example, where the secondchannel is reduced in depth relative to the first channel by one orderof magnitude, the pressure induced flow will be reduced 1000 times whilethe electroosmotically induced flow will be reduced by only a factor often. Accordingly, in some aspects, the present invention providesmicrofluidic devices as generally described herein, e.g., having atleast first and second intersecting channels disposed therein, but wherethe first channel is deeper than the second channel. Generally, thedepths of the channels may be varied to obtain optimal flow conditionsfor a desired application. As such, depending upon the application, thefirst channel may be greater than about two times as deep as the secondchannel, greater than about 5 times as deep as the second channel, andeven greater than about ten times as deep as the second channel.

[0070] In addition to their use in mitigating pressure effects, variedchannel depths may also be used to differentially flow fluids withindifferent channels of the same device, e.g., to mix differentproportions of fluids from different sources, and the like.

[0071] III. Electropipettor

[0072] As described above, any subject material can be transportedefficiently through the microfluidic system 100 in or near the subjectmaterial regions 200. With the first and second spacer regions 201 and202, the subject materials are localized as they travel through thechannels of the system. For efficient introduction of subject matterinto a microfluidic system, the present invention also provides anelectropipettor which introduces subject material into a microfluidicsystem in the same serial stream of combinations of subject materialregion 200, first and second spacer regions 201 and 202.

[0073] A. Structure and Operation

[0074] As illustrated in FIG. 4A, an electropipettor 250 is formed by ahollow capillary tube 251. The capillary tube 251 has a channel 254 withthe dimensions of the channels of the microfluidic system 100 to whichthe channel 254 is fluidly connected. As shown in FIG. 4A, the channel254 is a cylinder having a cross-sectional diameter in the range of1-100 μm, with a diameter of approximately 30 μm being preferable. Anelectrode 252 runs down the outside wall of the capillary tube 251 andterminates in a ring electrode 253 around the end of the tube 251. Todraw the subject materials in the subject material regions 200 with thebuffer regions 201 and 202 into the electropipettor channel 254, theelectrode 252 is driven to a voltage with respect to the voltage of atarget reservoir (not shown) which is fluidly connected to the channel254. The target reservoir is in the microfluidic system 100 so that thesubject material regions 200 and the buffer regions 201 and 202 alreadyin the channel 254 are transported serially from the electropipettorinto the system 100.

[0075] Procedurally, the capillary channel end of the electropipettor250 is placed into a source of subject material. A voltage is applied tothe electrode 252 with respect to an electrode in the target reservoir.The ring electrode 253, being placed in contact with the subjectmaterial source, electrically biases the source to create a voltage dropbetween the subject material source and the target reservoir. In effect,the subject material source and the target reservoir become Point A andB in a microfluidic system, i.e., as shown in FIG. 2A. The subjectmaterial is electrokinetically introduced into the capillary channel 254to create a subject material region 200. The voltage on the electrode252 is then turned off and the capillary channel end is placed into asource of buffer material of high ionic concentration. A voltage isagain applied to the electrode 252 with respect to the target reservoirelectrode such that the first spacer region 201 is electrokineticallyintroduced into the capillary channel 254 next to the subject materialregion 200. If a second or low ionic concentration spacer region 202 isthen desirable in the electropipettor channel 254, the end of thecapillary channel 254 is inserted into a source of low ionicconcentration buffer material and a voltage applied to the electrode252. The electropipettor 250 can then move to another source of subjectmaterial to create another subject material region 200 in the channel254.

[0076] By repeating the steps above, a plurality of subject materialregions 200 with different subject materials, which are separated byfirst and second spacer regions 201 and 202, are electrokineticallyintroduced into the capillary channel 254 and into the microfluidicsystem 100.

[0077] Note that if the sources of the subject material and the buffermaterials (of low and high ionic concentration) have their ownelectrode, the electrode 252 is not required. Voltages between thetarget reservoir and the source electrodes operate the electropipettor.Alternatively, the electrode 252 might be in fixed relationship with,but separated from, the capillary tube 251 so that when the end of thetube 251 contacts a reservoir, the electrode 252 also contacts thereservoir. Operation is the same as that described for the FIG. 4Aelectropipettor.

[0078]FIG. 4B illustrates a variation of the electropipettor 250 of FIG.4A. In this variation the electropipettor 270 is not required to travelbetween a subject material source and buffer material sources to createthe first and second spacer regions 201 and 202 within the pipettor. Theelectropipettor 270 has a body 271 with three capillary channels 274,275 and 276. The main channel 274 operates identically to the channel254 of the previously described electropipettor 250. However, the twoauxiliary capillary channels 275 and 276 at one end are fluidlyconnected to buffer source reservoirs (not shown) and the other end ofthe channels 275 and 276 is fluidly connected to the main channel 274.One reservoir (i.e., connected to auxiliary channel 275) holds buffermaterial of high ionic concentration, and the other reservoir (i.e.,connected to the channel 276) holds buffer material of low ionicconcentration.

[0079] All of the reservoirs are connected to electrodes forelectrically biasing these reservoirs for operation of theelectropipettor 270. The electropipettor 270 may also have an electrode272 along the walls of its body 271 which terminates in a ring electrode273 at the end of the main channel 274. By applying voltages on theelectrode 272 (and ring electrode 273) to create voltage drops along thechannels 274, 275, 276, not only can subject material be drawn into themain channel 274 from subject material sources, but buffer material ofhigh and low ionic concentrations can also be drawn from the auxiliarychannels 275 and 276 into the main channel 274.

[0080] To operate the electropipettor 270 with the electrode 272, theend of the main capillary channel 274 is placed into a source 280 ofsubject material. A voltage is applied to the electrode 272 with respectto an electrode in the target reservoir to create a voltage drop betweenthe subject material source 280 and the target reservoir. The subjectmaterial is electrokinetically drawn into the capillary channel 274. Thecapillary channel end is then removed from the subject material source280 and a voltage drop is created between the target reservoir connectedto the channel 274 and the reservoir connected to the channel 275. Afirst or high ionic strength spacer region 201 is formed in the channel274. Capillary action inhibits the introduction of air into the channel274 as the buffer material is drawn from the auxiliary channel 275. If asecond or low ionic concentration spacer region 202 is then desired inthe main channel 274, a voltage is applied to the electrodes in thetarget reservoir and in the reservoir of low ionic concentration buffermaterial. A second spacer region 202 is electrokinetically introducedinto the capillary channel 274 from the second auxiliary channel 276.The electropipettor 270 can then move to another source of subjectmaterial to create another subject material region 200 in the channel274.

[0081] By repeating the steps above, a plurality of subject materialregions 200 with different subject materials, which are separated byfirst and second spacer regions 201 and 202, are electrokineticallyintroduced into the capillary channel 274 and into the microfluidicsystem 100.

[0082] If it is undesirable to expose the subject material source tooxidation/reduction reactions from the ring electrode 273, theelectropipettor may be operated without the electrode 272. Becauseelectroosmotic flow is slower in solutions of higher ionic strength, theapplication of a potential (− to +) from the reservoir connecting thechannel 274 to the reservoir connecting the channel 275 results in theformation of a vacuum at the point where the channels 274 and 275intersect. This vacuum draws samples from the subject material sourceinto the channel 274. When operated in this mode, the subject materialis somewhat diluted with the solutions in the channels 275 and 276. Thisdilution can be mitigated by reducing the relative dimensions of thechannels 276 and 275 with respect to the channel 274.

[0083] To introduce first and second spacer regions 201 and 202 into thecapillary channel 274, the electropipettor 270 is operated as describedabove. The capillary channel end is removed from the subject materialsource 280 and a voltage drop is created between the target reservoirfor the channel 274 and the reservoir connected to the selected channels275 or 276.

[0084] Although generally described in terms of having two auxiliarychannels and a main channel, it will be appreciated that additionalauxiliary channels may also be provided to introduce additional fluids,buffers, diluents, reagents and the like, into the main channel.

[0085] As described above for intersecting channels within amicrofluidic device, e.g., chip, pressure differentials resulting fromdifferentially mobile fluids within the different pipettor channels alsocan affect the control of fluid flow within the pipettor channel.Accordingly, as described above, the various pipettor channels may alsobe provided having varied channel depths relative to each other, inorder to optimize fluid control.

[0086] B. Method of Electropipettor Manufacture

[0087] The electropipettor might be created from a hollow capillarytube, such as described with respect to FIG. 4A. For more complexstructures, however, the electropipettor is optimally formed from thesame substrate material as that of the microchannel system discussedabove. The electropipettor channels (and reservoirs) are formed in thesubstrate in the same manner as the microchannels for a microfluidicsystem and the channeled substrate is covered by a planar cover element,also described above. The edges of the substrate and cover element maythen be shaped to the proper horizontal dimensions of the pipettor,especially its end, as required. Techniques, such as etching, airabrasion (blasting a surface with particles and forced air), grindingand cutting, may be used. Electrodes are then created on the surface ofthe substrate and possibly cover, as required. Alternatively, the edgesof the substrate and the cover element may be shaped prior to beingattached together. This method of manufacture is particularly suited formultichannel electropipettors, such as described immediately above withrespect to FIG. 4B and described below with respect to FIG. 8.

[0088] IV. Sampling System

[0089] As described above, the methods, systems and apparatusesdescribed above will generally find widespread applicability in avariety of disciplines. For example, as noted previously, these methodsand systems may be particularly well suited to the task of highthroughput chemical screening in, e.g., drug discovery applications,such as is described in copending U.S. patent application Ser. No.08/671,987, filed Jun. 28, 1996, and previously incorporated byreference.

[0090] A. Sample Matrices

[0091] The pipetting and fluid transport systems of the invention aregenerally described in terms of sampling numbers of liquid samples,i.e., from multi-well plates. In many instances, however, the number ornature of the liquid based samples to be sampled may generate samplehandling problems. For example, in chemical screening or drug discoveryapplications, libraries of compounds for screening may number in thethousands or even the hundreds of thousands. As a result, such librarieswould require extremely large numbers of sample plates, which, even withthe aid of robotic systems, would create myriad difficulties in samplestorage, manipulation and identification. Further, in some cases,specific sample compounds may degrade, complex or otherwise possessrelatively short active half-lives when stored in liquid form. This canpotentially result in suspect results where samples are stored in liquidform for long periods prior to screening.

[0092] Accordingly, the present invention provides sampling systemswhich address these further problems, by providing the compounds to besampled in an immobilized format. By “immobilized format” is meant thatthe sample material is provided in a fixed position, either byincorporation within a fixed matrix, i.e., porous matrix, chargedmatrix, hydrophobic or hydrophilic matrix, which maintains the sample ina given location. Alternatively, such immobilized samples includesamples spotted and dried upon a given sample matrix. In preferredaspects, the compounds to be screened are provided on a sample matrix indried form. Typically, such sample matrices will include any of a numberof materials that can be used in the spotting or immobilization ofmaterials, including, e.g., membranes, such as cellulose,nitrocellulose, PVDF, nylon, polysulfone and the like. Typically,flexible sample matrices are preferred, to permit folding or rolling ofthe sample matrices which have large numbers of different samplecompounds immobilized thereon, for easy storage and handling.

[0093] Generally, samples may be applied to the sample matrix by any ofa number of well known methods. For example, sample libraries may bespotted on sheets of a sample matrix using robotic pipetting systemswhich allow for spotting of large numbers of compounds. Alternatively,the sample matrix may be treated to provide predefined areas for samplelocalization, e.g., indented wells, or hydrophilic regions surrounded byhydrophobic barriers, or hydrophobic regions surrounded by hydrophilicbarriers (e.g., where samples are originally in a hydrophobic solution),where spotted materials will be retained during the drying process. Suchtreatments then allow the use of more advanced sample applicationmethods, such as those described in U.S. Pat. No. 5,474,796, wherein apiezoelectric pump and nozzle system is used to direct liquid samples toa surface. Generally, however, the methods described in the '796 patentare concerned with the application of liquid samples on a surface forsubsequent reaction with additional liquid samples. However, thesemethods could be readily modified to provide dry spotted samples on asubstrate.

[0094] Other immobilization or spotting methods may be similarlyemployed. For example, where samples are stable in liquid form, samplematrices may include a porous layer, gel or other polymer material whichretain a liquid sample without allowing excess diffusion, evaporation orthe like, but permit withdrawal of at least a portion of the samplematerial, as desired. In order to draw a sample into the pipettor, thepipettor will free a portion of the sample from the matrix, e.g., bydissolving the matrix, ion exchange, dilution of the sample, and thelike.

[0095] B. Resolubilizing Pipettor

[0096] As noted, the sampling and fluid transport methods and systems ofthe present invention are readily applicable to screening, assaying orotherwise processing samples immobilized in these sample formats. Forexample, where sample materials are provided in a dried form on a samplematrix, the electropipetting system may be applied to the surface of thematrix. The electropipettor is then operated to expel a small volume ofliquid which solubilizes the previously dried sample on the matrixsurface (dissolves a retaining matrix, or elutes a sample from animmobilizing support), e.g., by reversing the polarity of the fieldapplied to the pipettor, or by applying a potential from the low ionicconcentration buffer reservoir to the high ionic concentration bufferreservoir, as described above. Once the sample is resolubilized, thepipettor is then operated in its typical forward format to draw thesolubilized sample into the pipettor channel as previously described.

[0097] A schematic illustration of one embodiment of an electropipettoruseful in performing this function, and its operation, is shown in FIG.8. Briefly, the top end 802 of the pipettor (as shown) 800 is generallyconnected to the assay system, e.g., a microfluidic chip, such thatvoltages can be supplied independently to the three channels of thepipettor 804, 806 and 808. Channels 804 and 808 are typically fluidlyconnected to buffer reservoirs containing low and high ionicconcentration fluids, respectively. In operation, the tip of thepipettor 810 is contacted to the surface of a sample matrix 812 where animmobilized (e.g., dried) sample 814 is located. A voltage is appliedfrom the low ionic concentration buffer channel 804 to the high ionicconcentration buffer channel 808, such that buffer is forced out of theend of the pipettor tip to contact and dissolve the sample. As shown,the pipettor tip 816 may include a recessed region or “sample cup” 818in order to maintain the expelled solution between the pipettor tip andthe matrix surface. In some cases, e.g., where organic samples are beingscreened, in order to ensure dissolution of the sample, an appropriateconcentration of an acceptable solvent, e.g., DMSO, may be included withthe low ionic concentration buffer. Voltage is then applied from thehigh ionic concentration buffer channel to the sample channel 806 todraw the sample into the pipettor in the form of a sample plug 820. Oncethe sample is completely withdrawn from the sample cup into thepipettor, the high surface tension resulting from air entering thesample channel will terminate aspiration of the sample, and high ionicconcentration buffer solution will begin flowing into the sample channelto form a first spacer region 822, following the sample. Low ionicconcentration buffer solution may then be injected into the samplechannel, i.e., as a second spacer region 824, by applying the voltagefrom the low ionic concentration buffer channel 804 to the samplechannel 806. Prior to or during presentation of the next sample positionon the matrix, a first or high ionic concentration spacer region 822 maybe introduced into the sample channel by applying the voltage betweenthe high ionic concentration buffer channel and the sample channel. Asnoted previously, a roll, sheet, plate, or numerous rolls, sheets orplates, of sample matrix having thousands or hundreds of thousands ofdifferent compounds to be screened, may be presented in this manner,allowing their serial screening in an appropriate apparatus or system.

[0098] V. Elimination of Electrophoretic Bias

[0099] As explained above, electrokinetic forces are used to transportthe subject material through the channels of the microfluidic system100. If the subject material is charged in solution, then it is subjectto not only electroosmotic forces, but also to electrophoretic forces.Thus the subject material is likely to have undergone electrophoresis intraveling from one point to another point along a channel of themicrofluidic system. Hence the mixture of the subject material or thelocalization of differently charged species in a subject material region200 at the starting point is likely to be different than the mixture orlocalization at the arrival point. Furthermore, there is the possibilitythat the subject material might not even be in the subject materialregion 200 at the arrival point, despite the first spacer regions 201.

[0100] Therefore, another aspect of the present invention compensatesfor electrophoretic bias as the subject materials are transportedthrough the microfluidic system 100. one way to compensate forelectrophoretic bias is illustrated in FIG. 5. In the microfluidicsystem 100 described above, each of the channels 110, 112, 114 and 116was considered as a unitary structure along its length. In FIG. 5, anexemplary channel 140 is divided into two portions 142 and 144. Thesidewalls of each channel portion 142 and 144 have surface charges whichare of opposite polarity. The two channel portions 142 and 144 arephysically connected together by a salt bridge 133, such as a glass fritor gel layer. While the salt bridge 133 separates the fluids in thechannel 140 from an ionic fluid in a reservoir 135. which is partiallydefined by the salt bridge 133, the salt bridge 133 permits ions to passthrough. Hence the reservoir 135 is in electrical, but not fluid,communication with the channel 140.

[0101] To impart electroosmotic and electrophoretic forces along thechannel 140 between points A and B, electrodes 132 and 134 are arrangedat points A and B respectively. Additionally, a third electrode 137 isplaced in the reservoir 135 at the junction of the two portions 142 and144. The electrodes 132 and 134 are maintained at the same voltage andthe electrode 137 at another voltage. In the example illustrated in FIG.5, the two electrodes 132 and 134 are at a negative voltage, while theelectrode 137 and hence the junction of the two portions 142 and 144 areat zero voltage, i.e., ground. Thus the voltage drops, and hence theelectric fields, in the portions 142 and 144 are directed in oppositedirections. Specifically, the electric fields point away from eachother. Thus the electrophoretic force on a particularly charged moleculeis in one direction in the channel portion 142 and in the oppositedirection in the channel portion 144. Any electrophoretic bias on asubject material is compensated for after traveling through the twoportions 142 and 144.

[0102] The electroosmotic force in both portions 142 and 144 are stillin the same direction, however. For example, assuming that the sidewallsof the channel portion 142 have positive surface charges, which attractnegative ions in solution, and the sidewalls of the channel portion 144have negative surface charges, which attract positive ions in solution,as shown in FIG. 5, the electroosmotic force in both portions 142 and144 is to the right of the drawing. Thus the subject material istransported from point A to point B under electroosmotic force, whilethe electrophoretic force is in one direction in one portion 142 and inthe opposite direction in the other portion 144.

[0103] To create a channel with sidewalls having positive or negativesurface charges, one or both portions of the channel is coated withinsulating film materials with surface charges, such as a polymer. Forexample, in the microfluidic system 100 the substrate 102 and thechannels may be formed of glass. A portion of each channel is coatedwith a polymer of opposite surface charge, such as polylysine, forexample, or is chemically modified with a silanizing agent containing anamino function, such as aminopropyltrichlorosilane, for example.Furthermore, the surface charge densities and volumes of both channelportions should be approximately the same to compensate forelectrophoretic bias.

[0104] Rather than being formed in a solid planar substrate, the channelcan also be formed by two capillary tubes which are butted together witha salt bridge which separates an ionic fluid reservoir from fluids inthe capillary tubes. An electrode is also placed in the ionic fluidreservoir. One capillary tube has a negative surface charge and theother capillary tube a positive surface charge. The resulting capillarychannel operates as described above.

[0105] FIGS. 6A-6D illustrates another embodiment of the presentinvention for which the effects of electrophoretic bias induced upon thesubject material is moving from point A to point B are compensated. Inthis embodiment the subject material is mixed at point B, anintersection between two channels, such as illustrated in FIG. 1.

[0106] FIGS. 6A-6D show a chamber 160 is formed at the intersection ofthe channels 150, 152, 154 and 156. The chamber 160 has four sidewalls162, 164, 166 and 168. The sidewall 162 connects a sidewall of thechannel 152 to a sidewall of the channel 150; the sidewall 164 connectsa sidewall of the channel 154 to the other sidewall of the channel 152;the sidewall 166 connects a sidewall of the channel 156 to the othersidewall of the channel 154; and the sidewall 168 connects the oppositesidewall of the channel 156 to the opposite sidewall of the channel 150.Assuming a flow of materials through the channel 152 toward the channel156, the sidewalls 162 and 168 form as a funnel if the materials arediverted into the channel 150.

[0107] The dimensions of the sidewalls 162 and 168 accommodate thelength of a subject material plug 200 traveling along the channel 152.The sidewalls 162 and 168 funnel down the plug 200 into the width of thechannel 150. The width of the channel 150 is such that diffusion of thesubject material occurs across the width of the channel 150, i.e.,mixing occurs and any electrophoretic bias of the subject materialcreated in the subject material region 200 traveling along the channel162 is eliminated. For example, if the channel 150 is 50 μm wide,diffusion across the channel occurs in approximately one second for amolecule having a diffusion constant of 1×10⁻⁵ cm²/sec.

[0108] In FIG. 6A a plug 200 of cationic subject material is movingalong the channel 152 towards the channel 156. By the time the plug 200reaches the chamber 160, the subject material has undergoneelectrophoresis so that the material is more concentrated at the forwardend of the subject material region 200. This is illustrated by FIG. 6B.Then the voltage drop impressed along the channels 152 and 156 isterminated and a voltage drop is created along the channels 154 and 150to draw the subject material region 200 into the channel 150. Thesidewalls 162 and 168 of the chamber 160 funnel the subject materialregion 200 with its electrophoretically biased subject material. This isillustrated by FIG. 6C. By diffusion the subject material is spreadacross the width of the channel 150 before the subject material travelsany significant distance along the channel 150; the subject material inthe subject material region 200 is mixed and ready for the next step ofoperation in the microfluidic system 100.

[0109] In addition to its use in correcting electrophoretic bias withina single sample, it will be appreciated that the structure shown in FIG.6 will be useful in mixing fluid elements within these microfluidicdevices, e.g., two distinct subject materials, buffers, reagents, etc.

EXAMPLES Example 1 Forced Co-Migration of Differentially ChargedSpecies, in Electropipettor-Type Format

[0110] In order to demonstrate the efficacy of methods used to eliminateor reduce electrophoretic bias, two oppositely charged species wereelectrokinetically pumped in a capillary channel, and comigrated in asingle sample plug. A Beckman Capillary Electrophoresis system was usedto model the electrophoretic forces in a capillary channel.

[0111] In brief, a sample containing benzylamine and benzoic acid ineither low ionic concentration (or “low salt”) (5 mM borate), or highionic concentration (“high salt”) (500 mM borate) buffer, pH 8.6, wasused in this experiment. The benzoic acid was present at approximately2× the concentration of the benzylamine. All injections were timed for0.17 minutes. Injection plug length was determined by the injectionvoltage, 8 or 30 kV. The low salt and high salt buffers were asdescribed above.

[0112] In a first experiment, three successive injections of sample inlow salt buffer were introduced into a capillary filled with low saltbuffer. Injections were performed at 8 kV and they were spaced by lowsalt injections at 30 kV. Data from these injections is shown in FIG.7A. These data show that benzylamine (identifiable as short peaks,resulting from its lesser concentration) from the first and secondinjections precede the benzoic acid peak (tall peak) from the firstinjection. Further, the benzylamine peak from the third injection isnearly coincident with the first benzoic acid peak. Thus, thisexperiment illustrates the effects of electrophoretic bias, whereinsample peaks may not exit a capillary channel in the same order theyentered the channel. As can be clearly seen, such separations cansubstantially interfere with characterization of a single sample, orworse, compromise previously or subsequently introduced samples.

[0113] In a second experiment, the capillary was filled with low saltbuffer. Samples were injected by first introducing/injecting high saltbuffer into the capillary at 8 kV (first spacer region 1). This wasfollowed by injection of the sample in high salt buffer at 8 kV, whichwas followed by a second injection of high salt buffer at 8 kV (firstspacer region 2). Three samples were injected in this manner, and spacedapart by injecting a low salt buffer at 30 kV. As can be seen in FIG.7B, both compounds contained in the sample were forced to co-migratethrough the capillary channel in the same sample plug, and arerepresented by a single peak from each injection. This demonstratessample registration, irrespective of electrophoretic mobility.

[0114] By reducing the size of the low salt spacer plug between thesamples, relative to the size of the samples, partial resolution of thecomponents of each sample injection can be accomplished. This may beuseful where some separation of a sample is desired duringelectrokinetic pumping, but without compromising subsequently orpreviously injected samples. This was carried out by injecting thelow-salt spacer plug at 8 kV rather than 30 kV. The data from thisexample is shown in FIG. 7C.

Example 2 Migration of Subject Materials through Electropipettor intoMicrofluidic System Substrate

[0115] FIGS. 9A-9C illustrate the experimental test results of theintroduction of a subject material, i.e., a sample, into a microfluidicsystem substrate through an electropipettor as described above. Thesample is rhodamine B in a phosphate buffered saline solution, pH 7.4. Ahigh ionic concentration (“high salt”) buffer was also formed from thephosphate buffered saline solution, pH 7.4. A low ionic concentration(“low salt”) buffer was formed from 5 mM Na borate, pH 8.6, solution.

[0116] In the tests subject material regions containing the fluorescentrhodamine B were periodically injected into the capillary channel of anelectropipettor which is joined to a microfluidic system substrate. Highsalt and low salt buffers were also injected between the subjectmaterial regions, as described previously. FIG. 9A is a plot of thefluorescence intensity of the rhodamine B monitored at a point along thecapillary channel near its junction with a channel of the substrateversus time. (In passing, it should be observed that the numbers onfluorescent intensity axis of the FIGS. 9A-9C and 10 plots are forpurposes of comparative reference, rather than absolute values.) Theinjection cycle time was 7 seconds and the electric field to move thesubject material regions through the electropipettor was 1000 volts/cm.The integration time for the photodiode monitoring light from thecapillary channel was set at 10 msec. From the FIG. 9A plot, it shouldbe readily evident that light intensity spikes appear at 7 secondintervals, matching the injection cycle time of the fluorescentrhodamine B.

[0117] In another experiment, the same buffers were used with therhodamine B samples. The monitoring point was in the channel of thesubstrate connected to the electropipettor. The injection cycle time wasset at 13.1 seconds and the voltage between the source reservoircontaining the rhodamine B and destination reservoir in the substrateset at −3000 volts. The monitoring photodiode integration time was 400msec. As shown in FIG. 9B, the fluorescent intensity spikes closelymatch the rhodamine B injection cycle time.

[0118] The results of a third experimental test are illustrated in FIG.9C. In this experiment the electropipettor was formed from a substrateand shaped by air abrasion. The monitoring point is along the capillarychannel formed in the substrate (and a planar cover). Here the samplematerial is 100 μM of rhodamine B in a buffer of PBS, pH 7.4. The highsalt buffer solution of PBS, pH 7.4, and a low salt buffer solution of 5mM Na borate, pH 8.6, are also used. Again periodic spikes offluorescent intensity match the cyclical injection of rhodamine B intothe electropipettor.

[0119]FIG. 10 illustrates the results of the cyclical injection ofanother subject material into an electropipettor, according to thepresent invention. In this experiment the sample was a small moleculecompound of 100 μM with 1% DMSO in a phosphate buffered saline solution,pH 7.4. A high salt buffer of the same phosphate buffered salinesolution, pH 7.4, and a low salt buffer of 5 mM Na borate, pH 8.6, wasalso used. The applied voltage to move the subject material regionsthrough the electropipettor was −4000 volts, and the integration timefor the photodiode monitoring light from the capillary channel was setat 400 msec. The samples were periodically injected into theelectropipettor as described previously. As in the previous results, theFIG. 10 plot illustrates that for small molecule compounds, theelectropipettor moves the samples at evenly spaced time (and space)intervals.

[0120] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques described abovemay be used in various combinations. All publications and patentdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication or patent document were so individually denoted.

What is claimed is:
 1. A microfluidic system with compensation forelectrophoretic bias, comprising a capillary channel having sides, afirst end and a second end, said capillary channel further divided intofirst and second portions, said sides of said first and second portionshaving surface charges of opposite polarities; and a first electrode atsaid first end; a second electrode between said first and secondportions of said capillary channel; and a third electrode at said secondend, said first, second and third electrodes set at voltages such that afluid is electroosmotically pumped through said first and secondportions from said first end to said second end, and electrophoreticmovement in said second portion is opposite to electrophoretic movementin said first portion.
 2. The microfluidic system of claim 1 whereinsaid first and second portions of said capillary channel each havesurface charge densities, said surface charge densities approximatelyequal.
 3. The microfluidic system of claim 2 wherein said first andsecond portions of said capillary channel each have volumes, saidvolumes approximately equal.
 4. The microfluidic system of claim 1wherein said sides of at least one of said portions of said capillarychannel are defined by a film, said film having said surface charge ofsaid one portion.
 5. A microfluidic system with compensation forelectrophoretic bias, comprising a first capillary channel; a secondcapillary channel intersecting said first capillary channel; and achamber at said intersection of said first and second capillary channelsshaped such that a region containing subject material moving from saidfirst capillary channel to said second capillary channel is mixed tocompensate for electrophoretic bias in moving along said first capillarychannel.
 6. The microfluidic system of claim 5 wherein said chamber isdefined by sides along a first capillary channel length funneling intosaid second capillary channel.
 7. The microfluidic system of claim 6wherein said chamber sides are straight.
 8. The microfluidic system ofclaim 6 wherein said second capillary channel width is approximatelyequal to a length of said subject material region.
 9. The microfluidicsystem of claim 5 wherein said subject material has a diffusion constantand said second capillary channel has a width such that said subjectmaterial diffuses across said second channel width before being divertedfrom said second channel.
 10. The microfluidic system of claim 9 whereinsaid subject material has a diffusion constant of approximately 1×10⁻⁵cm²/sec. and said second channel width is approximately 10 μm.
 11. Anelectropipettor for introducing materials into a microfluidic system,said electropipettor fluidly connected to said microfluidic system, saidelectropipettor comprising a capillary channel having an end forcontacting at least one source of said materials; and a voltage sourcefor applying a voltage between said one source of said materials and asecond electrode in said microfluidic system when said capillary channelend contacts said one source of said materials such that material fromsaid one source is electrokinetically introduced into saidelectropipettor toward said microfluidic system.
 12. The electropipettorof claim 11 wherein said capillary channel has a cross-sectional area ofapproximately 10-100 (μm)².
 13. The electropipettor of claim 11 whereinsaid voltage source applies a negative voltage to said first electrodewith respect to said second electrode.
 14. The electropipettor of claim11 wherein said capillary channel is defined in a substrate and by acover element over said substrate.
 15. An electropipettor forintroducing subject materials into a microfluidic system, saidelectropipettor fluidly connected to said microfluidic system, saidelectropipettor comprising a first capillary channel having a first endfor contacting at least one source of said subject materials and asecond end terminating in said microfluidic system; a second capillarychannel having a first end terminating near said first end of said firstcapillary channel and a second end terminating in a source of firstspacer material; a voltage source for applying voltages between said atleast one subject material source and said microfluidic system, andbetween said first spacer material source and said microfluidic systemsuch that subject material from said one subject material source andspacer material from said first spacer material source areelectrokinetically introduced into said electropipettor toward saidmicrofluidic system.
 16. The electropipettor of claim 15 furthercomprising a third capillary channel having a first end terminating nearsaid first end of said first capillary channel and a second endterminating in a source of second spacer material; and wherein saidvoltage source applies voltages to said second spacer material sourceand said microfluidic system such that subject material from said secondspacer material source is electrokinetically introduced into saidelectropipettor toward said microfluidic system.
 17. The electropipettorof claim 16 wherein said first spacer material source holds spacermaterial of ionic concentration orders of magnitude greater than that ofsaid second spacer material source.
 18. The electropipettor of claim 15further comprising an electrode disposed along said first capillarychannel such that said electrode contacts said subject material sourcewhen said first capillary channel end is placed into at least one sourceof said subject materials; and said voltage source is connected to saidelectrode to apply voltages between said at least one subject materialsource and said microfluidic system.
 19. The electropipettor of claim 15wherein said first and second capillary channels are defined in asubstrate and by a cover element over said substrate.
 20. A method ofintroducing materials from a plurality of sources into a microfluidicsystem, said microfluidic system having a capillary channel having anend, a voltage source for applying a voltage potential to an electrodein said microfluidic system, said method comprising contacting saidcapillary channel end to a subject material source; applying a voltageto said subject material source with respect to said electrode such thatsubject material from said source is electrokinetically introduced intosaid capillary channel toward said microfluidic system; selecting asource of spacer material, said spacer material having a predeterminedionic concentration; contacting said capillary channel end into saidsource of spacer material; applying a voltage to said spacer materialsource with respect to said electrode such that said spacer material iselectrokinetically introduced into said capillary channel next to saidsubject material; and repeating the steps above with different materialsources so that a plurality of different materials separated by spacermaterial is electrokinetically introduced into said capillary channeland transported toward said microfluidic system without intermixing saiddifferent materials.
 21. The method of claim 20 wherein said spacermaterial comprises a solution of high ionic strength.
 22. The method ofclaim 20 wherein said spacer material comprises a substantiallyimmiscible fluid.
 23. The method of claim 20 wherein said spacermaterial comprises an ionophore.
 24. The method of claim 20 wherein saidcapillary channel has a cross-sectional area of approximately 10-1000(μm)².
 25. The method of claim 20 wherein said steps of contacting saidcapillary channel end to a source of spacer material and applying avoltage to said spacer material source with respect to said firstelectrode to electrokinetically introduce said spacer material into saidcapillary channel, further comprise: placing said capillary channel endinto a source of a first spacer material; applying a voltage to saidfirst spacer material source with respect to said electrode such thatsaid first spacer material is electrokinetically introduced into saidcapillary channel; placing said capillary channel end into a source of asecond spacer material; applying a voltage to said second spacermaterial source with respect to said electrode such that said secondspacer material is electrokinetically introduced into said capillarychannel; and repeating said first two steps above so that said pluralityof different subject materials are separated by regions of said first,second and first spacer materials.
 26. The method of claim 25 whereinsaid first spacer material comprises a solution of high ionic strength,and said second spacer material comprises a solution of low ionicstrength.
 27. The method of claim 20 further comprising disposing asecond electrode along said capillary channel to said capillary channelend so that said second electrode contacts a source when said capillarychannel end contacts said material or spacer source; and wherein saidvoltage applying steps comprise creating a voltage difference betweensaid microfluidic system electrode and said second electrode.
 28. Themethod of claim 20 wherein said voltage applying steps comprise applyinga negative voltage to said subject material or spacer sources withrespect to said microfluidic system electrode.
 29. A microfluidic systemfor moving a plurality of subject materials from a first location to asecond location along a channel, said microfluidic system comprising: asource for creating a voltage difference across said first location andsaid second location; a plurality of subject material regions in saidchannel, said subject material regions separated by first spacer regionsof high ionic strength; and at least one second spacer region of lowionic concentration.
 30. A microfluidic system for transporting aplurality of subject material regions from a first point to a secondpoint on a channel, said microfluidic system comprising: a pair of firstspacer regions on either side of each subject material region, saidfirst spacer regions having high ionic concentrations; at least onesecond spacer region, said second spacer region having a low ionicconcentration.
 31. The microfluidic system of claim 30, wherein saidsecond spacer region has an ionic concentration at least two orders ofmagnitude less than that said first spacer region.
 32. The microfluidicsystem of claim 31 wherein said second spacer region has an ionicconcentration in the range of 1 to 10 mM, and said first spacer regionshave ionic concentrations in the range of 100 to 1000 mM.
 33. Amicrofluidic system, comprising: a substrate having at least a firstchannel and at least a second channel disposed in said substrate, saidat least second channel intersecting said first channel, wherein saidfirst channel is deeper than said second channel; and an electroosmoticfluid direction system.
 34. The microfluidic system of claim 33, whereinsaid at least first channel is at least twice as deep as said secondchannel.
 35. The microfluidic system of claim 33, wherein said at leastfirst channel is at least five times deeper than said second channel.36. The microfluidic system of claim 33, wherein said at least firstchannel is at least about ten times deeper than said second channel. 37.In an electroosmotic fluid direction system, a method of controllablydelivering a fluid stream along a first channel, wherein said firstchannel is intersected by at least a second channel, and wherein saidfluid stream comprises at least two fluid regions having different ionicstrengths, the method comprising, providing said first channel with agreater depth than said second channel.
 38. A method of transportingfluid samples within a microfluidic channel, comprising: introducing atleast a plug of a first fluid material having a first ionic strengthinto said channel; introducing at least a first sample fluid plug intosaid channel; introducing at least a second fluid material plug havingsaid first ionic strength into said channel; introducing at least athird fluid material plug having a second ionic strength, said secondionic strength being lower than said first ionic strength; and applyinga voltage across said channel.
 39. The method of claim 38, wherein saidsteps of introducing said at least first fluid material plug, said atleast sample fluid plug, said at least second fluid material plug andsaid at least third fluid material plug comprise: placing an end of saidchannel in contact with a source of said at least first fluid material,and applying a voltage from said source of said at least first fluidmaterial to said channel, whereby said first fluid material isintroduced into said channel; placing an end of said channel in contactwith a source of said at least first sample fluid and applying a voltagefrom said source of said at least first sample fluid to said channel,whereby said first sample fluid is introduced into said channel; placingan end of said channel in contact with a source of said at least secondfluid material and applying a voltage from said source of said at leastsecond fluid material to said channel, whereby said second fluidmaterial is introduced into said channel; and placing an end of saidchannel in contact with a source of said at least third fluid materialand applying a voltage from said source of said at least third fluidmaterial to said channel, whereby said third fluid material isintroduced into said channel.
 40. A sampling system comprising: anelectropipettor of claim 16; a sample substrate, said sample substratehaving a plurality of different samples immobilized thereon; and atranslation system for moving said electropipettor relative to saidsample substrate.
 41. The sampling system of claim 40, wherein saidfirst end of said first capillary channel and said first end of saidsecond capillary channel terminate in a fluid retention well at a tip ofsaid electropipettor.
 42. The sampling system of claim 40, wherein saidplurality of different samples are dried onto a surface of said samplesubstrate, and wherein said electropipettor is capable of expelling anamount of a fluid to resolubilize said sample on said sample substrate.43. The sampling system of claim 42, wherein said samples are applied tosaid sample substrate surface in a fluid form, and said substratesurface comprises a plurality of fluid localization regions.
 44. Thesampling system of claim 43, wherein said fluid localization regionscomprise relatively hydrophilic regions surrounded by relativelyhydrophobic regions.
 45. The sampling system of claim 43, wherein saidfluid localization regions comprise relatively hydrophobic regionssurrounded by relatively hydrophilic regions.
 46. The sampling system ofclaim 43, wherein said fluid localization regions comprise depressionson said surface of said sample substrate.
 47. The use of a substratehaving a channel, in transporting at least a first subject material fromat least a first location to a second location along said channel,utilising at least one region of low ionic concentration which istransported along said channel due to an applied voltage.
 48. A use ofclaim 47, in which the ionic concentration of said one region issubstantially lower than that of said subject material.
 49. A use ofclaim 47 or claim 48, wherein a plurality of subject materials aretransported, separated by high ionic concentration spacer regions. 50.The use of a substrate having a channel along which at least a firstsubject material may be transported, in electrophoretic biascompensation, said channel being divided into a first and a secondportion, in which the wall or walls of said channel are oppositelycharged, such that electrophoretic bias on said at least first subjectmaterial due to transportation in said first portion is substantiallycompensated for by electrophoretic bias due to transport in said secondportion.
 51. A use of claim 50 in which a first electrode is located ata remote end of said first portion, a second electrode is located at theintersection between said portions and a third electrode is located at aremote end of said second portion.
 52. A use of any of claims 47 to 51,in which said substrate is a microfluidic system.
 53. A use of any ofclaims 47 to 51, in which the substrate is an electropipettor.
 54. A useof claim 53, in which said electropipettor has a main channel fortransportation of said subject material and at least one further channelfluidly connected to said main channel from which a further material tobe transported along said main channel is obtained.
 55. A use of claim54, in which said further material is drawn into said main channel as abuffer region between each of a plurality of separate subject materials.56. The use of a microfluidic system having at least a first and asecond fluid channel which intersect, in optimising flow conditions, thechannels having different depths.
 57. A use of claim 56 in which onechannel is between 2 to 10 times deeper than the other channel.
 58. Theuse of a microfluidic system having a first channel and a second channelintersecting the first channel, in electrophoretic compensation, theintersection between said channels being shaped such that a fluid beingtransported along said first channel towards said second channel ismixed at said intersection and any electrophoretic bias in the fluid isdissipated.
 59. A microfluidic system comprising a substrate having achannel, in which at least a first subject material is transported fromat least a first location to a second location along said channel,utilizing at least one region of low ionic concentration which istransported along said channel due to an applied voltage.
 60. The systemof claim 59, in which the ionic concentration of said one region issubstantially lower than that of said subject material.
 61. A system ofclaim 59 or claim 60, wherein a plurality of subject materials aretransported, separated by high ionic concentration spacer regions.
 62. Asubstrate having a channel along which at least a first subject materialmay be transported, in electrophoretic bias compensation, said channelbeing divided into a first and a second portion, in which the wall orwalls of said channel are oppositely charged, such that electrophoreticbias on said at least first subject material due to transportation insaid first portion is substantially compensated for by electrophoreticbias due to transport in said second portion.
 63. A substrate as claimedin claim 62 in which a first electrode is located at a remote end ofsaid first portion, a second electrode is located at the intersectionbetween said portions and a third electrode is located at a remote endof said second portion.
 64. An electropipettor comprising a system asclaimed in any of claims 59 to 61, or a substrate as claimed in claims62 or claim
 63. 65. An electropipettor as claimed in claim 64, having amain channel for transportation of said subject material and at leastone further channel fluidly connected to said main channel from which afurther material to be transported along said main channel is obtained.66. An electropipettor as claimed in claim 65, in which said furthermaterial is drawn into said main channel as a buffer region between eachof a plurality of separate subject materials.
 67. A microfluidic systemhaving at least a first and a second fluid channel which intersect, saidchannels having different depths, in order to optimize flow conditions.68. A system as claimed in claim 67 in which one channel is between 2 to10 times deeper than the other channel.
 69. A microfluidic system havinga first channel and a second channel intersecting said first channel,the intersection between said channels being shaped such that a fluidbeing transported along said first channel towards said second channelis mixed at said intersection and any electrophoretic bias in said fluidis dissipated.